Antisense technology is an effective means for reducing the expression of one or more specific gene products and can therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications. The pharmacological activity of antisense oligonucleotides and oligonucleotide analogs, like other therapeutics, depends on a number of factors that influence the effective concentration of these agents at specific intracellular targets. One important factor for oligonucleotides is the stability of the species in the presence of nucleases. It is unlikely that unmodified oligonucleotides will be useful therapeutic agents because they are rapidly degraded by nucleases. Modification of oligonucleotides to render them resistant to nucleases therefore is greatly desired. Modifications to enhance the effectiveness of the antisense oligonucleotides have taken many forms. These modifications include sugar-phosphate backbone modifications, base ring modifications and sugar moiety modifications.
Modification of oligonucleotides to enhance nuclease resistance generally has taken place on the phosphorus atom of the sugar-phosphate backbone. Phosphorothioates, methyl phosphonates, phosphoramidates and phosphotriesters have been reported to confer various levels of nuclease resistance. Phosphate-modified oligonucleotides, however, generally have suffered from inferior hybridization properties. See, e.g., Cohen, J. S., ed. Oligonucleotides: Antisense Inhibitors of Gene Expression, (CRC Press, Inc., Boca Raton Fla., 1989).
Various dephosphono linkages (linkages without the phosphorus atom) modifications have been synthesized and studied for their antisense properties. Nonionic, achiral amide linkages (De Mesmaeker et al., Chem. Int. Ed. Engl. 1994, 33, 226-229; Just et al., Synlett 1994, 137-139) were disclosed. A full account of the synthesis and properties of the five isomeric amide modifications was described (De Mesmaeker el al., (1994) Novel Backbone Replacements for Oligonucleotides, In Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook Eds. ACS Symposium Series 580:24-39). Gogoi et al. presented the synthesis of thioacetamido nucleic acids (TANA) backbone and thermal stability studies with complementary DNA and RNA sequences (Gogoi et al., Chem. Commun., 2006, pp. 2373-2375).
Several nitrogen containing backbone modifications similar to the amides were evaluated as dimeric nucleosides (Sanghvi et al., Nucleosides Nucleotides 1997, 16, pp. 907-916). Peoc'h reported the synthesis of four methylene(methylimino) (MMI) linked oligodeoxyribonucleotide dimers modified at the T-position with fluoro and/or methoxy groups and their incorporation into different sequences (Peoc'h et al., Nucleosides, Nucleotides & Nucleic Acids, 23, pp. 411-438, 2004). Amino linkages have been synthesized and studied for enhanced cellular absorption (Saha el al., Tetrahedron Lett. 1993, 34, 6017-6020; De Mesmaeker el al., J. Bioorg. Med. Chem. Lett. 1994, 4, pp. 395-398; Caulfield et al, Bioorg. Med. Chem. Lett. 1993, 3, pp. 2771-2776). Other nitrogen containing backbones include oxime (Sanghvi et al., In Nucleosides and Nucleotides as Antitumor and Antiviral Agents; C. K. Chu and D. C. Baker Eds.: Plenum Press: New York, 1993, pp. 311-324), methyleneimino (ibid), methyleneoxy(methylimino) (MOMI) (ibid), methylene(dimethylhydrazo) (MDH) (Sanghvi et al., Collect. Czech. Chem. Commun. Special Issue 1993, 58, pp. 158-162), hydroxyl(methyliminomethylene) (HMIM) (Sanghvi et al., 11th IRT Nucleosides & Nucleotides, Leuven, Belgium, Sep. 7-11, 1994 (poster presentation)), carbamate (Dabkowski et al., J Chem. Soc. Perkin Trans. 1 1994, pp. 817-829), oxyamide linkage (Burgess et al., J. Chem. Soc. Chem. Commun. 1994, pp. 915-916), N-substituted guanidine (Vandendrissche et al., J. Chem. Soc. 1993, pp. 1567-1575; Pannecouque et al., Tetrahedron 1994, 50, 7231-7246), urea (Kutterer et al., Bioory. Med. Chem. Lett. 1994, 3, pp. 435-438) and thiourea linkages (Vandendrissche et al., J. Chem. Soc. 1993, pp. 1567-1575).
Synthesis of sulfur-containing backbone modifications, such as sulfonamide (McElroy et al., Bioorg. Med. Chem. Lett. 1994, 4, 1071-1076), sulfamoyl (Dewynter et al., Acad. Sci. 1992, 315, pp. 1675-1682), sulfonate (Huang et al., Synlett 1993, pp. 83-84), sulfide (Wang et al., Chin. Chem. Lett. 1993, 4, pp. 101-104; Huang et al., Synlett 1993, pp. 83-84; Kawai et al., Nucleic Acids Res. 1993, 21, pp. 1473-1479; Meng et al., J. Angew. Chem. Int. Ed. Engl. 1993, 32, pp. 729-731; Just el al. (1994), Synthesis and Hybridization Properties of DNA Oligomers Containing Sulfide-Linked Dinucleosides. In Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook Eds. ACS Symposium Series 580; (pp. 52-65)), and sulfone linkages (Just el al. (1994), Synthesis and Hybridization Properties of DNA Oligomers Containing Sulfide-Linked Dinucleosides. In Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook Eds. ACS Symposium Series 580; (pp. 62-65)) have been accomplished by several research groups.
Another backbone substitution is the formacetal and the related thioformacetal (Jones et al., J. Org. Chem., 58, pp. 2983-2991, 1993). Matteucci reported the synthesis of oligonucleotide analogs with one or more phosphodiester linkages that are replaced by a formacetal/ketal type linkage (U.S. Pat. No. 5,264,562 filed Apr. 24, 1991).
Chemically modified nucleosides are routinely used for incorporation into antisense sequences to improve the properties of antisense oligonucleotides as an alternative approach. One such group of chemical modifications includes bicyclic nucleosides wherein the furanose portion of the nucleoside includes a bridge connecting two atoms on the furanose ring thereby forming a bicyclic ring system. Such bicyclic nucleosides have various names including BNA's and LNA's for bicyclic nucleic acids or locked nucleic acids respectively.
Various BNA's have been prepared and reported in the patent literature as well as in scientific literature, see for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Wengel et al., PCT International Application WO 98-DK393 19980914; Singh et al., J. Org. Chem., 1998, 63, 10035-10039, the text of each is incorporated by reference herein, in their entirety. Examples of issued U.S. patents and published applications include for example: U.S. Pat. Nos. 7,053,207, 6,770,748, 6,268,490 and 6,794,499 and published U.S. applications 20040219565, 20040014959, 20030207841, 20040192918, 20030224377, 20040143114 and 20030082807; the text of each is incorporated by reference herein, in their entirety. Many LNA's are toxic. See, e.g., Swayze, E. E.; Siwkowski, A. M.; Wancewicz, E. V.; Migawa, M. T.; Wyrzykiewicz, T. K.; Hung, G.; Monia, B. P.; Bennett, C. F., Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals (Nucl. Acids Res., doi: 10.1093/nar/gkl1071, December 2006, advanced online publication).
Amide-linked dimers having one or two LNA nucleosides have been prepared and placed at internal positions within an oligomeric compound to determine their effects on Tm relative to a DNA/RNA duplex (Lauritsen et al., Chem. Commun., 2002, 530-532).
The present disclosure combines and incorporates both the synthesis of modified sugar-phosphate backbone and bicyclic nucleosides in attempting to enhance the properties of antisense oligonucleotides. Disclosed herein are neutral backbone bicyclic nucleic acid dimer analogs and antisense compounds prepared therefrom useful for modulating gene expression pathways, including those relying on mechanisms of action such as RNaseH, RNAi and dsRNA enzymes, as well as other antisense mechanisms based on target degradation or target occupancy. One having skill in the art, once armed with this disclosure will be able, without undue experimentation, to identify, prepare and exploit antisense compounds for these uses.